Lead by A. Morrie Craig and Anita Holman

History of the Oregon Seed & Straw Industries n : Field burning of grass straw phase-out completed u 250,000 acres down to 40,000 acres

History of the Oregon Seed & Straw Industries n Exportation of straw begins to increase u Pollution control tax credit from OR legislature u Export of grass straw to Japan grows to 250,000 tons/year in 1991 u Seed companies intensify use of endophyte since it lessens the need for pesticides, fertilizers and water F Pushes up concentration of ergot alkaloids in straw, creating the need for clinical-related research and testing and research for a long term, proactive solution

History of the Oregon Seed & Straw Industries n u Grass seed export increases over 200% (250,000 to 500,000 tons/year) F Represents 250,000 acres/year that are no longer burned u 1996: Oregon Legislature votes on money to be directed towards endophyte research at OSU

Crisis in Japan n 1998: shipments to Japan were shut down by Japanese gov’t d.t. fear of high level of endophyte toxins n Dr. Craig, seed industry and National Hay Association established the endophyte testing program and reaffirmed commitment to finding a long term solution u Reopened export to Japan n 1999-present: funding from the OR Ag Fiber Assn, state of Oregon and USDA was granted to study practical and clinical aspects of endophyte-infected forages and their effects on livestock n 2002: export increases to over 600,000 tons/year

Number of Test Requests Increasing ↑ Crisis in Japan

Tall fescue ~ Ergovaline HPLC Analysis Perennial ryegrass ~Lolitrem B HPLC Analysis

Threshold Levels for Clinical Disease (Tall fescue) Species Ergovaline (ppb) Horses* Cattle Sheep *Except for mares in the last 60 to 90 days of pregnancy, when the threshold is zero. when the threshold is zero.

Threshold Levels for Clinical Disease (Perennial ryegrass) Species Lolitrem B (ppb) Horses* Not determined Cattle Sheep *Except for mares in the last 60 to 90 days of pregnancy, when the threshold is zero. when the threshold is zero.

Sample Analysis Grind → Extract → Sample Prep HPLC → Chromatogram → Analysis and Data Reduction

Analysis of Endophyte Toxins n Log in samples u Accession number and submission form u Enter into computer log and case records

Analysis of Endophyte Toxins n Grind Samples u Use a grinding mill with 0.5 mm screen u Clean between each sample u Store in freezer until analysis

Analysis of Endophyte Toxins n Schedule run u Priority n Label glassware n Weigh out run u Duplicate F Samples F Controls F Reference material u 1.00g ergovaline, 0.200g lolitrem B

Analysis of Endophyte Toxins n Add mixing solutions u Ergovaline: 10 ml CHCl 3, 1 ml internal standard (ergotamine tartrate) and 1 ml N NaOH u Lolitrem B: CHCl 3 :MeOH (2:1 v/v) n Cap

Analysis of Endophyte Toxins n Turn in rotorack for hours

Analysis of Endophyte Toxins n Centrifuge samples n Pull out supernatant

Analysis of Endophyte Toxins n Extraction u Ergovaline: manifold with SPE (1.0g silica and 0.5g sodium sulfate) F Condition with CHCl 3 F Add 5 ml supernatant F Wash twice with 1 ml 75% CHCl 3 /25% acetone 75% CHCl 3 /25% acetone F Wash with 1.5 ml MeOH F Elute with 2.5 ml MeOH

Analysis of Endophyte Toxins n Concentrate u Ergovaline: dry eluent in N 2 evaporator at 50°C u Lolitrem B: dry 1.8 ml supernatant in N 2 evaporator n Reconstitute u Ergovaline: 0.5 ml MeOH u Lolitrem B: 0.5 ml CHCl 3 :ACN (4:1 v/v) n Vortex and sonicate n Final “filter” u Ergovaline: centrifuge u Lolitrem B: centrifuge in Nanosep microcentrifuge tubes microcentrifuge tubes u Add this solution to amber HPLC vials

Analysis of Endophyte Toxins n Run samples on HPLC system u Program computer and auto sampler F Create sequence and load method u Make mobile phase u Warm up system and prime pump u Load vials into auto sampler u Start injections

Analysis of Endophyte Toxins n Evaluate chromatograms u Draw baselines u Look for ‘abnormal’ fingerprint u Quantify

Analysis of Endophyte Toxins n Enter results into log file n Generate client summary and certificates u Fax of phone results u Send results and billing information n File lab copy of results n Transfer completed samples to long term storage freezer

Quality Control Program for the Tall Fescue and Perennial Ryegrass Toxins, Ergovaline and Lolitrem B Oregon State University College of Veterinary Medicine Dr. Craig’s Laboratory

Accurate and consistent results from the analysis of samples for ergovaline and lolitrem B between laboratories in Oregon, Missouri and Japan

Make the Perfect Standard

NMR of Ergovaline Synthesized by Dr. Forrest Smith in December DMSO H2OH2O

CHN Analysis of December Sample ExpectedFound C57.22%57.02% H6.11%6.33% N10.11%10.04% 2 Ergovaline + 2 Tartaric acid + H 2 O 2 Ergovaline + 2 Tartaric acid + H 2 O

Ergovaline Crystalline Standard OSU Method Ergovaline Ergotamine Ergovaline epimer Ergotamine epimer 2.8 µg/ml 5.6 µg/ml 1.12 µg/ml 0.28 µg/ml

New Quality Control Reference Material for Interlaboratory Analysis n Zero Seed n 300 ppb seed n 3300 ppb seed

Chuck Estill, Shanna Ivey, Anita Holman, Jennifer Duringer, A. Morrie Craig

The Problem n Reproductive problems at four dairies in the Willamette Valley in fall-winter 2003 u cows not coming into heat as frequently u silent heats u decreased conception rates

Causes n Viral or bacterial u Dr’s Estill and Holcheck involved → exams showed no sign of pathogen involvement n Mycotoxin u screened for two dairies → negative n Something else u same innoculum used for ensiling process by all four dairies u “new” toxin…

Causes con’t n Endophyte u prolactin: no obvious trend u ergovaline and lolitrem B: low levels in hay and silage u new peak: RT = 5 minutes in EV assay F only in silage, not hay from same field F another ergot alkaloid?

Chromatograms from Four Dairies

HPLC of Collected Peak

MS of 5 Minute Peak

MS/MS (m/z 593) of 5 Minute Peak Ergovaline mw = Molecular formula = C 29 H 35 N 5 O 5 mw = 60 C=12, O=16, H=1

Taking it one step further…MS 7 of 5-minute peak m/z minute peak m/z 533

Where do we go from here? n Identify the molecule u Nuclear Magnetic Resonance Spectroscopy F How many C and H? u C,H, & N Analysis F What percent is the molecule composed of C,H, & N? n Post-incident (2004…) u Reproduction problems abated u 5 minute peak has decreased in some cases, others it is still there